1. Field of the Invention
The present invention relates, in general, to a transposon. In particular, the present invention relates to a transposon consisting of a segment of the resistance (R) plasmid pCxM82B, test vectors containing this transposon, and methods of mutagenesis.
2. Background Information
Transposons contain a DNA sequence encoding a protein which makes it possible for the transposon to integrate non-specifically into the DNA of a chromosome or of a plasmid by means of illegitimate recombination. In addition, a transposon comprises a selectable marker (e.g. an antibiotic resistance gene). The presence of a selectable marker within the transposon aids in the identification and selection of cells containing this transposon. Therefore, it is possible to directly identify transposon-tagged integration mutants and determine the localization of the particular transposon-tagged gene in the genome. The isolation of a restriction fragment containing the particular gene and the inserted transposon can then take place in accordance with methods known in the art.
In contrast to transposon mutagenesis, traditional methods of mutagenesis (hydroxylamine mutagenesis and NTG mutagenesis) often negatively influence the vitality of the organism. Another problem associated with using traditional methods of mutagenesis to study genes is the inability to rapidly localize the mutated gene.
The technique of transposon mutagenesis is already being used successfully in gram-positive and in gram-negative bacteria. Note here by way of example the transposons Tn10 (9.3 kb, tetracycline resistance (Tc')) of plasmid R100 (Kleckner et al. (1981) Ann. Rev. Genet. 15, 341ff) and Tn5 (5.7 kb , kanamycin resistance (Km')) from the plasmid JR67 (Berg et al. (1975) Proc. Natl. Acad. Sci. USA 72, 3628ff), both from gram-negative bacteria, and the transposons Tn917 (5.1 Kb, Em') from the plasmid pAD2 (Tomich et al. (1980) J. Bact. 141, 1366ff) and Tn916 (15 kb, Tc') from the chromosome of Streptococcus faecalis DS16, both from gram-positive bacteria.
The transposon Tn917 was used successfully in bacillus ssp as well as in other gram-positive and gram-negative organisms (Kuramitsu and Casadaban (1986) J. Bact. 167, 711f) for generating auxotrophic cells mutant in amino-acid metabolism or in energy metabolism (Perkins and Youngman (1984) Plasmid 12, 119ff; Vandeyar and Zahler (1986) J. Bact. 167, 530ff; Mc Laughlin and Hughes (1989) J. Gen. Microbiol. 135, 2329).
In addition, transposons are presently being used for genome mapping, for the mobilization of replicons, for the generation of operon fusions and for the induction of genes (Simon et al. (1989) Biotechnology 1, Methods in Enzymology 118, 641-659).
Unfortunately, the known transposon-mutagenesis systems are not suitable for the mutating and genetic engineering of coryneform bacteria, especially those coryneform bacteria which produce and excrete amino acids.